The purpose of this proposal is to develop novel mouse models of colorectal cancer (CRC) with appropriate immune responses. These models have been designed to address limitations present in current models of CRC in order to enhance their suitability for translational research in immunotherapy. Immune checkpoint inhibitors have revolutionized treatment of solid tumors, and brought to light the critical importance of tumor immune context in treatment outcome. CRC with DNA mismatch repair (MMR) deficiency is characterized by a high burden of somatic mutations, increased T cell infiltration, and a favorable response to checkpoint blockade. Unfortunately, the majority of CRC has a lower mutational burden and is refractory to these treatments. Preclinical mouse models are powerful platforms for investigating the factors underlying response to immunotherapy. However, no single model faithfully recapitulates primary tumor development in the colon microenvironment, metastatic dissemination to the liver and lung, and the major immune contexts underlying variability in immunotherapy response in human CRC. To address these significant translational deficiencies, we will use an innovative technique employing colonoscopy-guided sub-mucosal injection of lentivirus or tumor organoids to induce focal autochthonous and orthotopic tumors in the colon that readily metastasize. In Aim 1, we will engineer a model that modulates tumor immunogenicity through inducible expression of a model antigen. We will dissect the features of the induced anti-tumor T cell response and investigate the utility of this model for testing adoptive T cell therapy by transferring antigen-specific T cells. To potentiate adoptive T cell therapy and mirror ongoing clinical trials in humans, we will assess the efficacy of CRISPR-Cas9-mediated deletion of immune checkpoints in T cells prior to transfer. In Aim 2, we will model immunotherapy-responsive CRC by targeting the essential DNA MMR genes Msh2 and Mlh1 and use next-generation sequencing to characterize the mutational landscapes of resulting MMR-deficient versus proficient tumors. In Aim 3, we will perform preclinical trials of immune checkpoint blockade in these models to explore their ability to recapitulate the responses of human CRC patient populations. We will also test a novel combination of immunogenic chemotherapy and checkpoint blockade, based on the hypothesis that immunogenic cell death may sensitize tumors with low mutational burden or minimal pre-existing T cell involvement to immune attack. This strategy is aimed at improving treatment for the majority of CRC patients, whose tumors are non-immunogenic and non- responsive to immunotherapy. The overarching goal of this research plan is to develop and benchmark a set of highly comparable CRC models that will be used to address why only a fraction of patients respond to immunotherapy. The proposed strategy is innovative in that it uses cutting-edge methods in mouse genetic engineering and cancer modeling to capture critical features of human CRC. This research will also include deep characterization of the immune microenvironment in these models and a comparison to humans.